植物中天然乙酰胆碱酯酶抑制剂的高通量发现研究

项目名称： 植物中天然乙酰胆碱酯酶抑制剂的高通量发现研究

项目编号： No.20802031

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 杨中铎

作者单位： 兰州理工大学

项目金额： 18万元

中文摘要： 本项目通过研究新型酶反应底物、显色剂以及改进现有方法的手段，建立了两种高通量、低费用的薄层色谱生物自显影方法用于天然乙酰胆碱酯酶抑制剂的识别和跟踪分离研究。新方法解决了现有方法中存在的酶用量大、薄层背景不明显不利于观察的问题。具有费用低、灵敏度高的优点。利用新方法，对常用中药、藏药和西北分布较广的贝母属、锦鸡儿属、杜鹃属植物以及植物内生菌中存在的天然乙酰胆碱酯酶抑制剂进行快速寻找和跟踪分离，发现了7个较高活性的先导化合物（2-羟基-1-甲氧基阿朴啡、geissoschizine methyl ether、茵芋碱、康丝碱，锥丝明，康丝瑞明，锥丝亚胺）。对部分活性化合物进行了与酶的计算机分子模拟对接研究。研究了其中的一个先导物-荷叶碱（2-羟基-1-甲氧基阿朴啡衍生物）的构效关系。三年来共发表论文20篇，其中SCI收录10篇；申请国家发明专利3项，其中2项已授权。本项目是快速寻找天然先导物的新思路，也为今后开展其它类酶抑制剂的研究提供了借鉴。建立的新薄层色谱生物自显影方法，为研究经费不足的研究者从事乙酰胆碱酯酶抑制剂的发现研究提供了捷径。

中文关键词： 薄层色谱生物自显影; 乙酰胆碱酯酶抑制剂; 高通量; 生物碱

英文摘要： Two high-throughput, low-cost thin-layer chromatographic (TLC) bioautographic methods for screening natural acetylcholinesterase (AchE) inhibitors from plants have been established by studying new substrate of the enzyme reaction, new chromogenic agent and improving existing methods. The problems of the high consumption of enzyme and light-colored background of TLC plate in documented methods have been solved in new methods. New methods have low-cost, high sensitive advantages. Seven high active lead compounds (2-hydroxy-1-methoxy aporphine, geissoschizine methyl ether, skimmianine, conessine, conessimine, conarrhimine, conimine) have been obtained by bioactivity-guided chromatographic fractionation of total alkaloidal extracts of Traditional Chinese medicine, Tibetan medicine and extracts of Fritillaria, Caragana ,Rhododendron genus. Valid docking program was applied to predict the possible binding mode of some lead compounds with AchE. The structure-activity relationship of nuciferine (one of the new-found lead compounds) have also been studied. In the past three years, a total of 20 papers, including 10 SCI have been published and three national invention patents have been applied, two of which have been authorized. This project is a new idea for quickly find lead compounds from natural resources. The successful experiences obtained from this project may be helpful for constructing new TLC-bioautographic methods for screening other enzyme inhibitors. Our new TLC-bioautographic methods will be benefit to research groups lacking enough funds pursuing for natural acetylcholinesterase inhibitors.

英文关键词： thin-layer chromatographic bioautographic method;acetylcholinesterase inhibitors; high-throughput;alkaloid